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In-vitro maturation of camel oocytes using different media and
sera
A. E. B. Zeidan1, M. E. Hammad2, Sh. A. Gabr2, M. H. Farouk3*, Sh. M. Shamiah1, E. A. A. Ahmadi1 and W. M. A. Nagy1.
1 Dept. Camel Res., Anim. Prod. Res. Instit., Dokki, Giza, Egypt. 2 Dept. Anim. Prod., Fac. Agric., Tanta Univ., Tanta, Egypt. 3 Dept. Anim. Prod., Fac. Agric., Al-Azhar Univ., Nasr City, Cairo, Egypt. *mhfarouk@azhar.edu.eg
Why?
• Milk
• Meat Economic role of dromedary camel
Therapeutic effect (Knoess, 1984; Abdalla et al., 2015).
• Male (Allam et al., 2013).
• Female (Shalash, 1980; Kandil et al., 2014). Seasonality of reproduction
Selection (Abdalla et al., 2015).
Culture medium (Humblot et al., 2005; Harris and Picton, 2007).
Very few data (Kandil et al., 2014).
Objective
Breeding & non-breeding season
Two maturation media
• TCM-199
• MEM
Three types of serum
• FDCS
• BSA
• ESS
In-vitro maturation of camel oocyte
Material and methods
Ovaries collection
Meteorological data
Laboratory
Ovaries collection
175 non-pregnant
she-camel
350 ovaries
110 during breeding season
240 during non-breeding
season
Meteorological data
Ta (°C) RH (%)
THI Day light
length (hr)
Laboratory
Harvesting media (PBS)
Maturation media
Oocyte collection
Oocyte categories
Fixation
Staining
Examination for maturation
Statistical analysis
Oocyte categories
Compact cumulus oocyte
• Oocyte with five or more layers of complete cumulus cells.
Partial denuded oocyte
• Oocyte with cumulus cells present either incompletely surrounding the oocyte.
Denuded oocyte
• Oocyte without cumulus cells and covered by zona pellucida.
Shrunken (degenerated) oocyte
• Ooplasm shrunken away from the zona pellucida or not evenly filling the zona ooplasm looks degenerated with fragment empty zona pellucida.
Photomicrograph of the different oocyte categories.
Compact cumulus
Partially denuded
Denuded oocyte
Degenerated oocyte
Treatment of maturation medium
• T1: TCM-199 + 10% FDCS.
• T2: TCM-199 + 6mg/ ml BSA.
• T3: TCM-199 + 10% ESS.
• T4: MEM + 10% FDCS.
• T5: MEM + 6mg/ml BSA.
• T6: MEM + 10% ESS.
The supplementation
included the following media:
Maturation
pH
• 7.2-7.4
osmolarity
• 280-300 mOsmol/kg
Filter
• 0.22 µm-millipore
maturation medium
• 200 µl
Time
• 40 hr
Temperature
• 38.5 °C
RH
• 90-95%
Table 1. Compositions of maturation media and sera
Amount
Per 100 ml Content
9 ml
9 ml
Media:
TCM-199 or
MEM (liquid)
10%
6 mg/ml
10%
Sera:
FDCS
BSA
ESS
20 µg/ml PMSG
10 IU/ml HCG
1 µg/ml Oestradiol-17β
20 mMol Na Pyruvate
100 IU/ml Na Penicillin G
100 µg/ml Streptomycin
Criteria of maturation
Germinal vesicle (GV)
• Inter phase chromosomes enclosed within a nuclear membrane.
Germinal vesicle breakdown (GVBD)
• An absence of a visible nuclear membrane and chromatin condensation characterized by a cluster of DNA material without individual chromosomes.
Metaphase-І (MІ)
• Chromosomes were condensed in pairs and without detected polar body (immature oocytes).
Metaphase-II (MII)
• One the larger group of chromosome formed an equatorial plate and the remaining chromosomes are highly condensed or had extruded a polar body (oocytes mature).
Degenerated
• Oocytes were vacuolated or had scattered or highly condensed chromatin.
Results
Table 2. Air temperature, relative humidity, temperature-humidity index and length of day light, during breeding and non-breeding seasons.
Season
Air temperature (⁰C) Relative humidity (%) Temperature-humidity
index (THI) Length of
day light
(hr)
Minimum Maximum Minimum Maximum Minimum Maximum
Breeding 11.23 21.66 43.02 58.49 50.80 68.02 12.24
Non-
breeding 20.84 34.30 42.67 63.66 65.88 86.60 15.24
Table 3. Effect of breeding and non-breeding season on in-vitro maturation rate of camel oocytes.
Season
Total
Oocyte
number
Criteria of maturation
Germinal vesicle Germinal vesicle
breakdown Metaphase-I Metaphase-II Degenerated
N % N % N % N % No. %
Breeding 710 99 13.9 93 13.1 120 16.9 257 36.2a 141 19.9b
Non-
breeding 650 86 13.2 90 13.9 111 17.1 217 33.4b 146 22.5a
a and b: Means denoted within the same column with different superscripts are significantly different (P<0.05).
Table 4. Effect of maturation media on in-vitro maturation rate of camel oocytes.
Type of
medium
Total Oocyte
number
Criteria of maturation
GV GVB Metaphase-І Metaphase-II Degenerated
N % N % N % N % N %
TCM 199 700 87 12.4 b 90 12.9 b 116 16.7 b 253 36.1a 154 22.0 a
MEM 660 98 14.8a 93 14.1a 115 17.4a 221 33.5 b 133 20.1 b
a and b: Means denoted within the same column with different superscripts are significantly different (P<0.05). TCM199 = Tissue Culture Medium; MEM = Minimum Essential Medium
Table 5. Effect of type of serum on in-vitro maturation rate of camel oocytes.
Type of
serum
Total oocyte
number
Criteria of maturation
Germinal vesicle Germinal vesicle
breakdown Metaphase-І
Metaphase-
II Degenerated
N % N % N % N % N %
FDCS 465 52 11.2b 61 13.1a 70 15.1b 190 40.8a 92 19.8b
BSA 455 64 14.1a 65 14.3a 83 18.2a 152 33.4b 91 20.0b
ESS 440 69 15.7a 57 13.0a 78 17.7a 132 30.0C 104 23.6a
a,b and C: Means denoted within the same column with different superscripts are significantly different at P<0.05. FDCS = Fetal Dromedary Camel Serum; BSA = Bovine Serum Albumin; ESS = Estrus Sheep Serum.
a and b: Means denoted within the same column with different superscripts are significantly different (P<0.05). FDCS = Fetal Dromedary Camel Serum; BSA = Boivne Serum Albumin; ESS = Estrus Sheep Serum
23, 10%
30, 12%
35, 15%
101; 42%
51, 21%
TCM 199 * FDCS
GV
GVB
Metaphase І
Metaphase II
Degenerated
28, 12%
33, 14%
41, 18%
80;
35%
48, 21%
TCM 199 * BSA
36, 16%
27, 12%
40, 17% 72;
31%
55, 24%
TCM 199 * ESS
29, 13%
31, 14%
35, 15% 89;
40%
41, 18%
MEM * FDCS
36, 16%
32, 14%
42, 19% 72;
32%
43, 19%
MEM * BSA
33, 16%
30, 14%
38, 18% 60;
29%
49, 23%
MEM * ESS
Conclusion
use of TCM-199 medium for in vitro maturation of camel oocytes added with fetal dromedary camel serum (FDCS) to promote in vitro camel oocytes maturation for in-vitro fertilization programme to enhance of fertilizing ability of she-camel oocytes, especially during breeding season.
Thank you for your attention
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